1. Field of the Invention
The present invention relates to a method for producing a cross-linked hyaluronic acid, especially producing cross-linking hyaluronic acid with decreased cross-linking agent content.
2. Description of the Prior Arts
Hyaluronic acid is a kind of polysaccharides, which is composed of disaccharides and 400 D of molecular weight. The disaccharide is composed of β-1,4-glucuronic acid and β-1,3-N-acetylglucosamine linked together by β-1,4 glycosidic bond. Moreover, the disaccharide links other disaccharides by β-1,3 glycosidic bonds to form linear polysaccharide. Currently, hyaluronic acid is synthesized by bacteria such as streptococcus and is obtained by extracted from animal tissues such as from cockscomb.
Since hyaluronic acid, hyaluronate and derivatives thereof have good biocompatibility, bio-degradability and viscoelasticity, they can be used in cosmetics, biomedicine, medical products and pharmaceutical industry.
Since the organism of linear hyaluronic acid can be easily degraded by enzymes, such as hyaluronidase and free radicals, residence time of the organism of linear hyaluronic acids is relevantly shorter. Furthermore, linear hyaluronic acid has low mechanical strength, so the linear hyaluronic acid has limited applications. Therefore, the cross-linked hyaluronic acid is most preferably used.
When practicing the operating method, the cross-linked hyaluronic acid have different type to the different goal, such as a solution type, a hydrogel type, a matter between a solution type and hydrogel type, and a mixture consisting of a solution type and a hydrogel type. A manufacture method for producing the cross-linked hyaluronic acid comprises: mixing a cross-linking agent and hyaluronic acid to proceed a cross-linking reaction and to obtain the cross-linked hyaluronic acid; and purifying the cross-linked hyaluronic acid to remove the excess cross-linking agent. The step of purifying the cross-linked hyaluronic acid may be by dialysis or washing with water or a buffer solution. However, the above-mentioned dialysis or washing cannot remove all of the residual cross-linking agent, especially because it has a bonding-state end and a free-state end. Furthermore, the free-state end of the cross-linking agent is still active and can create undesirable side effects when applied in animals, as shown by U.S. Pat. No. 5,808,050.
Also, the manufacture method for purifying the cross-linked hyaluronic acid by dialysis or washing also faces the following difficulties:
1. Refining cannot be easily scaled up at an industry level.
2. The cross-linked hyaluronic acid requires sterile conditions; otherwise, contaminants can easily be integrated into a final product. However, the manufacture method for the cross-linked hyaluronic acid by dialysis or washing is under neutral or almost neutral condition, so sterile conditions are almost always difficult to be controlled.
3. When the cross-linked hyaluronic acid is hydrogel and when the cross-linked hyaluronic acid with low degree of cross-linking swells significantly, the cross-linked hyaluronic acid is difficult to be washed and the cross-linked hyaluronic acid may be easily lost during the washing process. Similarly, when the cross-linked hyaluronic acid with high degree of cross-linking swells insignificantly, the cross-linking agent may not be capable of being removed with ease.
4. Moreover, the cross-linked hyaluronic acid with low degree of cross-linking can be easily washed away when washing the cross-linked hyaluronic acid, so that the residual cross-linked hyaluronic acid has decreased its lubrication. Therefore, when the cross-linked hyaluronic acid is used for injection, straight-chain or cross-linked hyaluronic acid solution has to be added in the residual cross-linked hyaluronic acid to increase its lubrication.
U.S. Pat. No. 4,716,154 discloses a manufacture method for a gel of cross-linked hyaluronic acid for use as a vitreous humor substitute, comprising: cross-Inking a hyaluronate with multifunctional cross-linking agent at 50° C. for two hours under alkaline condition and placing overnight at room temperature to obtain a gel; for removing un-reacted cross-linking agent, cutting the gel into small pieces and washing thoroughly for 24-hours using distilled water and further washing for 8 hours by boiled saline water to obtain gel with 0.23% to 1.2% of solid content. However, the foregoing method has the following shortcomings:
(1) a complex purifying step is required;
(2) the gel can swell and the gel content is low when removing cross-linking agent with boiled saline water and attaining a gel form. Therefore, the process needs extra step to improve the gel content; and
(3) the cross-linked hyaluronic acid has to swell again in the buffer solution to adjust the osmotic pressure and the pH value so that the method is unsuitable to be conducted at an industry level.
US 2006/0194758A1 discloses cross-linking of low and high molecular weight hyaluronic acids to obtain monophase hydrogels. The monophase hydrogels have good mechanical strength with improved properties for injection. However, the monophase hydrogels have residual cross-linking agent over 300 ppm after cross linking reaction at 50° C. Then, the residual cross-linking agent is intended to be removed by dialysis but the cross-linking agent cannot be removed thoroughly. Therefore, neither un-reacted cross-linking agent with two free-state ends nor reacted cross-linking agent with one free-state end and one bonding-state end in the hydrogels can be removed by dialysis or washing.
US 2005/0281880A1 discloses methods for making injecTable polymer hydrogels, comprising: (a) cross-linking one or more polymers to form a gel, (b) washing the gel, (c) purifying the gel, and (d) homogenizing the gel to produce the hydrogel. The method uses a di-functional or multifunctional cross-linking agent with high concentration to manufacture hydrogel. Therefore, the above-mentioned hydrogel contains residual cross-linking agents. Moreover, the hydrogel needs 2 to 3 days to wash and to purify the gel. However, the hydrogel is under almost neutral condition and may be polluted by microorganism.
US 2007/0026070A1 discloses a method for manufacturing a cross-linked polysaccharide composition comprising: (a) contacting a polysaccharide mixed in an alkaline medium with a bifunctional or polyfunctional epoxide to provide an essentially epoxy cross-linked polysaccharide, wherein the epoxide is substantially linked to the polysaccharide by ether bonds; (b) drying the epoxy cross-linked polysaccharide without substantially removing epoxide from the alkaline medium to form a cross-linked polysaccharide matrix; (c) optionally washing the cross-linked polysaccharide matrix with a water miscible solvent; and (d) neutralising the cross-linked polysaccharide matrix with an acidic medium to form a cross-linked polysaccharide gel. Therefore, the method delivers a product having residual cross-linking agents.
U.S. Pat. No. 4,716,224 discloses a method of manufacturing cross-linked hyaluronic acid using precipitation to remove the cross-linking agent. However, the method produces a product that still contains residual cross-linking agents.
When cross-linking polymers to form a gel in an alkaline medium, the cross-linking reaction competes with a hydrolysis reaction as shown by Y. Tokita and A Okamoto, “Hydrolytic Degration of Hyaluronic Acid”, Polymer Degration and Stability, vol. 48, pp. 269-273 (1995). In the initial reaction period, the concentration of the cross-linking agent is high, so the cross-linking reaction is the principal reaction. But, after a specific amount of the cross-linking agent is consumed, the hydrolysis reaction becomes the principal reaction and destroys the gel causing gel degradation. Therefore, the cross-linking reaction in an alkaline medium has to be terminated before such secondary reaction occurs, despite the fact that the gel still contains much residual cross-linking agent. The manufacturing method comprises: cross-linking a hyaluronate and di-functional or multi-functional cross-linking agent in 25 to 60° C. for a period of between 10 minutes and 24 hours under basic condition to obtain a gel; and purifying the gel to remove cross-linking agents. However, the cross-linking agent cannot be removed thoroughly. An actual reaction time depends on the temperature and the basic condition. No matter how the gel obtained in foregoing methods intend to achieve purification, a certain/significant amount of cross-linking agents still remains in the cross-linked hyaluronic acid.
WO-00/46253 A1 discloses a process for the production of multiple cross-linked hyaluronic acid derives. The process comprises two cross-linking reactions at an uniform temperature, and the cross-linking agents, feeding ratios, and/or pH values of the two cross-linking reactions are different. However, the cross-linking agents with free-state functional group in the cross-linked hyaluronic acid obtained thereby is quite high and the cross-linked hyaluronic acid cannot be applied to a human or an animal directly. A washing step and/or a purifying step is necessary for obtaining a physiologically acceptable cross-linked hyaluronic acid where the residual cross-linking agent in the product is over 300 ppm.
A preparation of cross-linked hyaluronic acid is also disclosed by Tomihata and Ikada (Biomaterials, 1997, 18, 189-195). A strategy to remove a cross-linking agent with free-state functional group provided is elongation of the reaction time. However, a washing step and/or a purifying step is necessary for obtaining a physiologically acceptable cross-linked hyaluronic acid where the residual cross-linking agent in the product is over 300 ppm. Furthermore, a deterioration in the cross-linked hyaluronic acid occurs that causes the cross-linked hyaluronic acid to turn deep yellow or deep brown or cause a final product to degrade obviously.
To overcome the shortcomings, the present invention provides a method for producing a cross-linked hyaluronic acid to mitigate or to obviate the aforementioned problems.